Synthesis of Amino-Functionalized Abasic Monomers as Versatile Building Blocks for siRNA Modification

Abstract Chemical modification plays a central role in optimizing siRNA therapeutics. Here, we designed and synthesized four amino-functionalized abasic phosphoramidite monomers (Ab1−Ab4) based on tetrahydropyrrole and aminocyclopentane scaffolds. Incorporation of these units destabilized siRNA duplexes in a position-dependent manner, and enhanced silencing activity at central or terminal positions on the sense strand, with the (S)-aminocyclopentane abasic (Ab4) exhibiting the strongest potency. Seed-region incorporation, particularly of the (R)-aminocyclopentane abasic (Ab3), mitigated miRNA-like off-target effects while retaining on-target activity in vitro. Following subcutaneous administration in mice, an Ab4-modified siRNA induced more potent hepatic mRNA knockdown than the parent siRNA at a single dose of 0.1 mg/kg, and Ab3 incorporation significantly reduced the number of off-target transcripts. In addition, 3′-terminal Ab4 incorporation improved nuclease resistance, leading to enhanced hepatic exposure in vivo, and served as a practical linker for conjugation. These findings establish amino-functionalized abasic units as a versatile platform for siRNA modification.